Fatty Acid Metabolism
The fatty acid biosynthetic pathway in man is comprised of four major enzymes: acetyl-CoA carboxylase, the rate limiting enzyme which synthesizes malonyl-CoA; malic enzyme, which produces NADPH; citrate lyase, which synthesizes acetyl-CoA; and fatty acid synthase, which catalyzes NADPH-dependent synthesis of fatty acids from acetyl-CoA and malonyl-CoA. The final products of fatty acid synthase are free fatty acids which require separate enzymatic derivatization with coenzyme-A for incorporation into other products. In man, significant fatty acid synthesis may occur in two sites: the liver, where palmitic acid is the predominant product (Roncari, Can. J. Biochem., 52:221-230, 1974); and lactating mammary gland where C.sub.10 -C.sub.14 fatty acids predominate (Thompson, et al., Pediatr. Res., 19: 139-143, 1985). Except for lactation, and cycling endometrium (Joyeux, et al., J. Clin. Endocrinol. Metab., 70:1319-1324, 1990), the fatty acid biosynthetic pathway is of minor physiologic importance, since exogenous dietary fatty acid intake down-regulates the pathway in the liver and other organs (Weiss, et al., Biol. Chem. Hoppe-Seyler, 367:905-912, 1986).
In liver, acetyl-CoA carboxylase, malic enzyme and fatty acid synthase are induced in concert by thyroid hormone and insulin via transcriptional activation and repressed by glucagon (Goodridge, Fed Proc., 45:2399-2405, 1986) and fatty acid ingestion (Blake, et al., J. Nutr., 120:1727-1729, 1990). Tumor necrosis factor alpha (TNF) a cytokine with profound effects on lipogenesis, is either stimulatory or inhibitory depending on the cell type studied. TNF markedly inhibits lipogenesis in adipocytes by reduction in acetyl-CoA carboxylase and fatty acid synthase protein synthesis, but is markedly stimulatory in the liver by increasing the level of citrate, which is the primary allosteric activator of the rate limiting enzyme of fatty acid biosynthesis, acetyl-CoA-carboxylase.
In lactating breast, the other major site of fatty acid biosynthesis in humans, fatty acid synthesis is under control of prolactin, estrogen, and progesterone. During pregnancy, progesterone acts as a mitogen to promote breast development and concomitantly down-regulates prolactin receptors, preventing lipid and milk protein synthesis before delivery. After delivery, the fall in estrogen and progesterone levels allows up-regulation of prolactin receptors and subsequent increase in lipogenic enzymes and milk protein production by breast epithelial cells.
Regulation of fatty acid synthase expression in human breast cancer has been studied primarily as a model for progesterone-stimulated gene expression. In contrast to normal lactating breast where progesterone stimulates epithelial cell growth while retarding lipogenic enzyme synthesis, in progesterone receptor (PR) positive human breast carcinomas such as MCF-7, ZR-75-1, and T-47D, progesterone inhibits growth and induces fatty acid synthase production along with other lipogenic enzymes (Chambon, et al., J. Steroid Biochem., 33:915-922 (1989). Progesterone presumably acts to up-regulate fatty acid synthase expression via the steroid hormone response element as is found in the rat fatty acid synthase promoter (Amy, et al., Biochem. J., 271:675-686, 1989), leading to increased FAS mRNA transcription or, by other mechanisms, to increased message stability (Joyeux, et al., Mol. Endrocinol., 4:681-686, 1989). Regarding PR-negative human breast cancer cells, a single study reports that fatty acid synthase accounts for about 25% of cytosolic protein in SKBR3 cells but no data regarding its biologic significance or regulation was available (Thompson, et al., Biochim. Biophys. Acta, 662:125-130, 1981).
With regard to cytokines and other lipogenic hormones, only scant data are available concerning human breast cancer. For example, TNF has been known to be markedly growth inhibitory to some breast cancer cultures. While TNF is mildly growth inhibitory to primary rat hepatocyte cultures (ID.sub.50 =5000 units/ml), some human breast cancer cells such as MCF-7 are extremely growth inhibited (ID.sub.50 =40 units/ml) (Chapekar, et al., Exp. Cell. Res., 185:247-257, 1989). The effect of TNF on FAS expression or lipogenic activity in breast cancer cells, however, remains unknown. One study of fatty acid synthase expression in MCF-7 cells using Northern analysis, found that insulin and insulin growth factor-1 were only slightly stimulatory compared to 5-10 fold increases seen with progesterone, while T.sub.3 had no effect (Chalbos, et al., J. Steroid Biochem. Molec. Bid., 43:223-228, 1992). Overall, regulation of FAS in receptor positive breast cancer has been only cursorily examined, while receptor negative tumors have not been studied.
No association with poor clinical outcome was found for breast or for any other cancers in those few systems where fatty acid synthase expression was studied. In the only study purporting to associate FAS expression with prognosis, fatty acid synthase expression was studied by in situ hybridization in 27 breast cancers, finding an association between increased fatty acid synthase mRNA and a higher degree of morphologic differentiation, but without association with estrogen or progesterone receptor status (Chalbos, et al., J. Natl. Cancer Inst., 82:602-606, 1990). It was deduced from these data that fatty acid synthase expression in breast carcinoma is associated with greater degree of morphologic differentiation and therefore presumably with less aggressive tumors. A second study of 87 cases by Northern blotting of fatty acid synthase mRNA found an association of fatty acid synthase expression and young age (premenopausal patients), but again no association with receptor status (Wysocki, et al., Anticancer Res., 10:1549-1552, 1990). Neither study provided clinical follow-up of their patients; there were no data comparing FAS expression with either disease-free interval or patient survival. Without clinical outcome, no reliable conclusions can be drawn regarding FAS expression and tumor virulence.
These studies stand in contrast to a series of greater than 200 patients from several centers demonstrating a strong association between poor prognosis and expression of a protein of undetermined function (designated OA-519) through measurement of disease-free survival or overall survival (Kuhajda, N. Engl. J. Med., 321:636-641, 1989; Shurbaji, et al., Am. J. Clin. Pathol, 96:238-242, 1991; Corrigan, et al., Am. J. Clin. Pathol., 96:406, 1991; Cote, et al., Lab. Invest. ,66:13A, 1992; Ziegler, et al., Am. J. Clin. Oncol., 14: 101-110, 1991).
Nor has fatty acid metabolism been a target of study in cancer therapeutics. Fujii, et al. (1986, Japan J. Exp. Med., 56:99-106), used the fatty acid synthase inhibitor cerulenin in combination with exogenous antitumor antibodies to weaken the cell membrane in an attempt to potentiate complement-mediated cell membrane damage via the membrane attack complex. Cerulenin was known to be toxic to cells at high concentration, and Fujii, et al., taught that the cerulenin concentration should be kept low to maintain the selectivity conferred by the humoral immune component of complement-mediated cell lysis. Spielvogel, et al., U.S. Pat. No. 5,143,907, noted that a series of phosphite-borane compounds exhibited both antineoplastic activity and anti-inflammatory activity while lowering serum cholesterol and serum triglycerides. The phosphite-borane compounds are non-specific inhibitors that affect many cellular functions, and so they are not selectively effective against tumor cells. Spielvogel, et al. taught that the hypolipidemic effect on serum cholesterol and triglycerides was mediated through more than one mechanism, and the antineoplastic effect was not shown to be related to the hypolipidemic activity.
Cerulenin is a potent inhibitor of fatty acid biosynthesis at the level of the FAS complex (S. Omura, Bacteriological Reviews, September 1976, p. 681-697, Vol. 40 No. 3). The structure of cerulenin and the mechanism of action are shown below: ##STR1## The alkylation of the critical enzyme thiol inactivates the Beta-ketoacyl-ACP synthetase component of the FAS multienzyme complex. Cerulenin may be viewed as having an enzyme binding moiety (the nine carbon diene) and a reactive group (the keto epoxy amide).
Cerulenin was originally isolated as a potential antifungal antibiotic from the culture broth of Cephalosporium caerulens. Structurally cerulenin has been characterized as 2R,3S-epoxy-4-oxo-7,10-trans,trans-dodecanoic acid amide. Its mechanism of action has been shown to be inhibition, through irreversible binding, of .beta.-ketoacyl-ACP synthase, the condensing enzyme required for the biosynthesis of fatty acids. Cerulenin has been categorized as an antifungal, primarily against Candida and Saccharomyces sp. In addition, some in vitro activity has been shown against some bacteria, actinomycetes, and mycobacteria, although no activity was found against Mycobacterium tuberculosis. The activity of fatty acid synthesis inhibitors and cerulenin in particular has not been evaluated against protozoa such as Toxoplasma gondii or other infectious eucaryotic pathogens such as Pneumocystis carinii, Giardia lamblia, Plasmodium sp., Trichomonas vaginalis, Cryptosporidium, Trypanosoma, Leishmania, and Schistosoma.
Despite cerulenin's in vitro activity against some bacteria and fungi it has not been developed as a therapeutic agent. To date research on this compound has centered on it use as a research tool for investigating the role of fatty acids in the metabolism and physiology of a variety of organisms because of its activity as a fatty acid synthesis inhibitor.